IgG-binding protein from Staphylococcus and nucleotide sequence encoding this protein

ABSTRACT

A recombinant DNA molecule coding for a protein expressed by a  Staphylococcus aureus  bacterium, comprising the nucleotide sequence SEQ ID NO:1 or a homologous sequence, or a partial or homologous sequence of SEQ ID NO:1 coding for a polypeptide fragment comprising at least 15 amino acid residues, is described. Further, a protein expressed by such a bacterium or a polypeptide fragment comprising at least 15 amino acid residues, comprising the amino acid sequence SEQ ID NO:2 binds IgG and apolipoprotein H. Examples of the polypeptide fragments comprise the SEQ ID NO:3 through 6. These proteins and polypeptide fragments may be coupled to an inert carrier or matrix. Vectors comprising such a DNA molecule or the corresponding RNA molecule, and antibodies specifically binding to a polypeptide having an amino acid sequence of SEQ ID NO:4 or SEQ ID NO:6, are also disclosed. The DNA or RNA molecules, the vectors and the antibodies mentioned may all be used in different types of vaccines against Staphylococcal infections. Moreover, a method of isolating and/or purifying apolipoprotein H from a liquid medium, especially from serum, is described.

The present invention relates to a new protein and a nucleotide sequenceencoding said protein. More precisely, the invention relates to a DNAmolecule coding for a protein expressed by a bacterium of the genusStaphylococcus aureus, said protein and polypeptide fragments of saidprotein. Vectors comprising the nucleotide sequence coding for theprotein, the protein and fragments thereof, and antibodies specificallybinding to the protein may all be used for different vaccines againstStaphylococcal infections in mammals. The invention also relates to amethod of isolating and/or purifying apolipoprotein H from e.g serumwith an immobilised protein or polypeptide of the invention.

BACKGROUND OF THE INVENTION

Staphylococcus aureus is a pathogen responsible for a wide variety ofdiseases in humans and animals, including endocarditis, osteomyelitis,wound sepsis and mastitis. The bacterium produces several potentialvirulence factors such as alpha-, beta-, gamma- and delta-toxins, toxicshock syndrome toxin (TSST), enterotoxins, leucocidin, proteases,coagulase and clumping factor.

It is generally accepted that adhesion to tissues is required forbacterial colonisation to occur. For this purpose staphylococci expresssurface adhesins, which interact with host matrix proteins such asfibronectin, vitronectin, collagen, laminin and bone sialoprotein. Inaddition, staphylococci are able to bind several serum proteins, such asIgG, fibronectin, fibrinogen, and thrombospondin, possibly masking thebacteria from the immune system of the host. However, the contributionand importance of each of these binding functions in differentinfections is still unclear.

The most studied receptor in S. aureus is protein A, a cellwall-associated protein, which binds to the Fc- and the Fab-regions ofIgG from several species. Protein A in strain 83254 consists of fiveconsecutive, highly homologous domains, all with IgG-binding activity,followed by a region anchoring the protein in the cell wall (Uhlén etal, 1984). IgG-binding ability is common among clinical strains of S.aureus suggesting an important function in pathogenesis. It has beenassumed that the IgG-binding capacity is mediated by protein A only.

However, the present inventors recently identified a nucleotide sequencein S. aureus strain 8325-4 encoding a polypeptide, clearlydistinguishable from protein A, which binds IgG in a non-immune fashion(Jacobsson & Frykberg, 1995). An IgG-binding protein fragment having anamino acid sequence of 84 aa was disclosed. However, the amino acidsequence of the full length protein and the properties other than theIgG-binding ability of the 84 aa fragment were not known or evensuggested. No nucleotide sequence coding for said protein has bedisclosed or suggested prior to the present invention.

Diseases caused by Staphylococcal infections are often treated withantibiotics. As is well known in the art, these microorganisms candevelop antibiotic resistance. Therefore, the use of vaccines to preventor contain the spread of infection would be desirable. At present, thereis no vaccine on the market that gives full protection. The presentinvention provides new immunologically active components for theproduction of vaccines against Staphylococcal infections.

DESCRIPTION OF THE INVENTION

The present invention is based on cloning and nucleotide sequencedetermination of a complete gene (sbi) encoding a novel IgG-bindingprotein. The gene encodes a protein of 436 amino acids, denoted proteinSbi, with one IgG-binding domain that exhibits an immunoglobulin-bindingspecificity similar to protein A and without the typical Gram-positivecell wall anchoring sequence LPXTG (SEQ ID NO:7) (Schneewind et al,1995) suggesting that the protein is not anchored in the cell wall.Analysis of other S. aureus strains shows that this gene is not uniquefor strain 8325-4. For instance, the Sbi-protein is highly expressed instrain Newman 4, which shows that the IgG-binding activity observed inS. aureus is not mediated only by protein A. In fact, this (sbi) gene ispresent in all tested strains of S. aureus.

Further, it has now been revealed that the Sbi protein of the inventionbinds apolipoprotein H, a major serum component, in addition to IgG.Hitherto, no bacterial protein binding to apolipoprotein H has beenreported. Therefore, neither is this combination of the protein bindingto these two serum components previously known. The portion of theprotein which binds to IgG is located near the N- terminal of theprotein, whereas the middle portion binds to apolipoprotein H. Thisenables the use of the protein, or an appropriate polypeptide fragment,in immobilised form for the isolation and/or purification ofapolipoprotein H.

Thus, one aspect of the present invention is directed to a recombinantDNA molecule coding for a protein expressed by a bacterium of the genusStaphylococcus aureus, comprising the nucleotide sequence SEQ ID NO:1,defined in the sequence listing and the claims, or a homologous sequenceto SEQ ID NO:1coding for said protein, or a partial or homologoussequence of the sequence SEQ ID NO:1 coding for a polypeptide fragmentof said protein comprising at least 15 amino acid residues.

This recombinant DNA molecule may be inserted into plasmids, phages orphagemides for the expression/production of the protein or proteinfragments.

Another aspect of the invention is directed to a protein expressed by abacterium of the genus Staphylococcus aureus or a polypeptide fragmentof said protein comprising at least 15 amino acid residues other thanthe 84 aa fragment at the position 38-121, which protein comprises theamino acid sequence SEQ ID NO:2, defined in the sequence listing and theclaims, or a homologous sequence to the sequence SEQ ID NO:2 comprisinga few mismatches in the amino acid sequence of SEQ ID NO:2, orpolypeptide fragments of said homologous sequence comprising at least 15amino acid residues.

The disclaimer of the 84 aa fragment at the position 38-121 of the SEQID NO:2 is made because, as already mentioned, it has been previouslydisclosed (Jacobsson & Frykberg, 1995).

It is well known in the art that there may be a few mismatches of aminoacids residues in the amino acid sequence of a protein while the proteinstill retains its major characteristics. The mismatches may bereplacements of one or several amino acids, deletions of amino acidresidues or truncations of the protein. Such mismatches occur frequentlyin genetic variations of native proteins. It is believed that up to 15%of the amino acid residues may be replaced in a protein while theprotein still retains its major characteristics. The protein of theinvention comprises 436 amino acid residues, and therefore up to 66mismatches would be acceptable. However, preferably there will be lessthan 20, more preferably less than 10, and most preferably less than 5mitsmatches in the amino acid sequence of the protein of the invention.

The polypeptide fragments of the protein of the invention shouldcomprise at least 15 amino acid residues to be sure that the fragmentsare not found in other known proteins. These fragments may be used e.g.as probes, diagnostic antigens, and vaccine components, possibly coupledto carriers.

In an embodiment of this aspect of the invention a polypeptide fragmentof the protein according to the invention has the amino acid sequenceSEQ ID NO:3, defined in the sequence listing and the claims. Thispolypeptide fragment lacks the signal sequence of the SEQ ID NO:1.

In another embodiment a polypeptide fragment of the protein according tothe invention has an amino acid sequence SEQ ID NO:4, defined in thesequence listing and the claims. This polypeptide fragment bindsapolipoprotein H.

In yet another embodiment a polypeptide fragment of the proteinaccording to the invention has the amino acid sequence SEQ ID NO:5,defined in the sequence listing and the claims. This 120 aa polypeptidefragment binds IgG. It was chosen for immunisation purposes, in stead ofthe known IgG binding 84 aa fragment, since once the whole amino acidsequence was deduced, it became evident there were sequence similaritiessuggesting two IgG binding domains.

In still another embodiment a polypeptide fragment of the proteinaccording to the invention has the amino acid sequence SEQ ID NO:6. Thispolypeptide fragment binds apolipoprotein H, and has been used forisolation and purification of said serum protein.

In a preferred embodiment of this aspect of the invention the protein orpolypeptide according to the invention is coupled to an inert carrier ormatrix. The carrier may be e.g. plastic surfaces, such as microplates,beads etc.; organic molecules such as biotin; proteins, such as bovineserum albumin; peptide linkers, polypeptides e.g. resulting in fusionproteins. The matrix may be particles used for chromatographic purposes,such as Sepharose®.

A further aspect of the invention is directed to a vector selected fromthe group consisting of plasmids, phages or phagemides comprising anucleotide sequence according to the invention.

These vectors may be used for the production of the proteins orpolypeptides of the invention. They may also be used in vaccines.

Yet another aspect of the invention is directed to antibodiesspecifically binding to a polypeptide having an amino acid sequenceselected from the group consisting of SEQ ID NO:4, and SEQ ID NO:6. Thespecific binding of binding of an antibody to an amino acid sequence ofthe invention requires e.g an affinity constant of at least 10⁷liters/mole, preferably at least 10⁹ liters/mole.

The antibodies of the invention may be monoclonal or polyclonal. Theymay be used in diagnostic tests, but preferably in vaccines for passiveimmunization.

Still another aspect of the invention is directed to the use of aprotein or polypeptide according to the invention, optionally inimmobilised form, as an immunising component in the production of avaccine against Staphylococcus infections.

Another use aspect of the invention is directed to the use of a vectoraccording to the invention for the production of a vaccine againstStaphylococcal infections.

Yet another use aspect of the invention is directed to the use ofantibodies according to the invention for the production of a vaccinefor the passive immunisation of a mammal against Staphylococcusinfections.

An additional aspect of the invention is directed to a vaccine againstStaphylococcal infections comprising as an immunising component aprotein or polypeptide according to the invention, optionally inimmobilised form.

Another vaccine aspect of the invention is directed to a vaccine againstStaphylococcal infections comprising a vector according to theinvention.

A DNA molecule, or the corresponding RNA derived from the presentsequence, as described in claim 1, may be used in a vector for vaccinepurposes. Examples of suitable forms of administration includeintravenous, percutaneous, and intramuscular administration.

Yet another vaccine aspect of the invention is directed to a vaccine forthe passive immunisation of a mammal, especially a human being, againstStaphylococcus infections comprising antibodies according to theinvention.

One embodiment of the invention comprises the passive immunization ofpatients with an impaired immune defense or patient awaiting majorsurgery, such as patients in line for an organ transplantation orawaiting the insertion of a prosthetic device, such as a hip prosthesisor similar major surgical intervention. According to the presentinvention, a high dose of antibodies against the novel protein can beadministered to any patient before or at the time of hospitalisation, inorder to prevent Staphylococcus infection.

The vaccines may contain other ingredients selected with regard to theintended administration rout, and these ingredients are chosen by thevaccine manufacturer in collabaration with pharmacologists. Examples ofadministration routs include intravenous administration, percutaneousadministration, oral and nasal administration.

A further aspect of the invention is directed to a method ofprophylactic and/or therapeutic treatment of Staphylococcus infectionsin a mammal comprising administration to said mammal of animmunologically effective amount of a vaccine according to any one ofthe vaccines of the invention.

Still another aspect of the invention is directed to a method ofisolating and/or purifying apolipoprotein H from a liquid medium,especially from serum, comprising chromatographic separation ofapolipoprotein H from said liquid medium with an immobilised protein orpolypeptide according to the invention as stationary phase.

In a preferred embodiment of the invention column chromatography is usedfor the isolation/purification of apolipoprotein H from blood serum. Theprotein or polypeptide of the invention is coupled to e.g. Sepharose®and is used as packing material for the column. The apolipoproteinH-containing serum is brought into contact with the immobilized proteinor polypeptide and the apolipoprotein H is adsorbed. Finally, theapolipoprotein H is eluated from the column.

DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a) the cloned 3 kb PstI-XbaI fragment from S. aureus strain8325-4 containing the sbi-gene of the invention (clone pPX1), and b)schematic drawing of the protein Sbi aligned with the peptides encodedby the phagemid clones isolated by panning against IgG. Stars indicatethat the clone was isolated several times independently. Ig4 representsthe clone isolated earlier (Jacobsson & Frykberg, 1995). S denotes thesignal sequence and the shaded bar represents a proline-rich region.

FIGS. 2a+2 b show the complete nucleotide sequence of the sbi-gene (SEQID NO:1) of the invention from S. aureus 8325-4, and the deduced aminoacid sequence of the encoded protein. Features of the sequence areindicated as follows: underbar, putative promotor sequences; doubleunderbar, possible ribosomal binding site; overbar, putativetranscriptional stop signal (inverted repeat); single vertical arrowindicates the cleavage site of the signal sequence and in bold therepeated proline residues. Also shown in bold is the minimal IgG-binding domain as deduced by shot gun phage display mapping.

FIG. 3 shows the specificity of the binding domain of the protein Sbi(clone Ig4) for various IgGs and human IgG3κ, IgG3λ, IgM and IgA. Eachcolumn represents a mean value of two independent experiments with thesame phage stock. The y-axis shows the number of c.f.u. in 50 μl of theeluate.

EXPERIMENTS

Analysis of IgG binding activity in S. aureus by Western blots usuallyreveals more than one protein band, which interact with IgG, and it hasbeen assumed that these polypeptides represent breakdown products ofprotein A. In view of the present results, it is not surprising thatprotein Sbi has escaped detection. For example, analysis of theexpression of the protein Sbi in S. aureus has been hampered by theability of protein A to interact with IgGs from most mammalian species.Furthermore, both proteins migrate similarly in SDS-PAGE and have thesame specificity for all tested immunoglobulins. As disclosed in Example4 below, the commercially available anti-protein A antibodiescross-react with full-length protein Sbi but not with MAL-SbiΔ (SEQ IDNO:4) (lacking the known IgG-binding domain). Most likely, thecross-reaction is not unique to this source of antibodies and may havecontributed to the failure to identify protein Sbi. Now the antibodiesdirected against the protein Sbi of the invention produced in chickenallows discrimination between expression of protein A and protein Sbi.

The present inventors have cloned and sequenced the sbi-gene from S.aureus strain 8325-4. The detection of protein Sbi, expressed from itsown promoter in S. epidermidis and as a MAL-fusion in E.coli withHRP-labelled IgG, proves that this gene encodes a second IgG-bindingprotein. This protein consists of 436 amino acids (SEQ ID NO:2) andcontains a signal sequence but lacks the cell wall sorting LPXTG (SEQ IDNO:7) motif, suggesting that the protein is not anchored in thecell-wall. However, in strain Newman 4, which produces high amounts ofprotein Sbi, no protein Sbi is detected in the culture growth medium(data not shown). Instead, protein Sbi is released from the cell surfaceby addition of sample buffer (Example 4), in contrast, release ofprotein A requires treatment with lysostaphin (Example 4). Thisindicates that protein Sbi is associated to the cell surface by adifferent mechanism. There are also other examples of cell surfaceassociated proteins that lack the LPXTG motif, such as the S. aureuselastin-binding protein (Park et al 1996) and MHC class II analogue(Jönsson et al 1995).

Mapping of protein Sbi by shot-gun phage display strongly suggests thatthe protein has only one IgG-binding domain with a deduced minimalbinding domain of 52 amino acids. Furthermore, expression and analysisof NH₂-terminally truncated Sbi-protein, i.e. the C-terminal part of theprotein, consisting of aa 143-436 ( SEQ ID NO:4), shows that noIgG-binding activity is located in this part of the protein (Example 4).The IgG-binding domain shows a significant homology to the IgG-bindingrepeats of protein A (FIG. 3).

A phage-stock made from the originally isolated clone, Ig4 (aa 38-121)(FIG. 1b), was used in an analysis of the immunoglobulin speciesreactivity (FIG. 3). A comparison between data on the specificity ofprotein A (Boyle, 1990) and the results for clone Ig4 (FIG. 3) shows,that the two proteins exhibit a very similar Ig-binding profile. Inaddition, a study using one or two binding domains from protein A(clones Ig7 (domain C) and Ig1 (D-A) described by Jacobsson & Frykberg(1995)) gave an Ig-binding profile similar to that of Ig4 (data notshown). The display of protein domain(s) on the phage surface offers aquick and sensitive method for analysing specific binding to othermolecules.

Further, the present protein has been shown to bind to another serumprotein, apolipoprotein H, also known as β2-glycoprotein I. Hitherto, nobacterial protein binding to apolipoprotein H has been reported. Thisfinding is unexpected and opens new possibilities to use the protein forisolation/purification of apolipoprotein H for research laboratories andfor the production of antibodies against the apolipoprotein H bindingportion of the protein. These antibodies may be used as components invaccines for passive immunisation of mammals against Staphylococcusinfections.

EXAMPLES Bacterial Strains Growth Conditions, Vectors and Helper Phage

The bacterial strains used are listed in Table 1. Phage R408 (Promega)was used as the helper phage for production of phage stocks. E. colicontaining the pUC18 or pMAL-c2 vectors (New England Biolabs) wereselected on LA-plates (Luria-Bertani (LB)-broth with 1.5% agar and 50 μgampicillin ml⁻¹) and grown in LB-broth supplied with 50 μg ampicillinml⁻¹ . E. coli containing the phagemid vector pHEN1 (Hoogenboom et al,1991), were grown in the same medium supplemented with 1% glucose (w/v).Staphylococcal strains were grown in Tryptone Soya Broth (TSB) (Oxoid).S. epidermidis containing pBR473 was grown in the same medium containing20 μg ml⁻¹ of chloramphenicol.

TABLE 1 Bacterial strains Species Strain Characteristics and use E. coliTG1 F⁺ and amber suppressing. Used for construction of the phage libraryand production of phage stocks. MC1061 Used for all other DNAmanipulations. S. aureus 8325-4 NCTC 8325 cured from prophages Wood 46Protein A-negative reference strain Newman 4 Spontanteous mutant ofstrain Newman with enhanced production of fibronectin-binding proteinCowan I NCTC 8350, high level producer of cell-wall-bound protein A. S.epidermidis 247

Example 1 Cloning and Sequencing of the sbi Gene Encoding an IgG-bindingProtein

Restriction and modification enzymes were purchased from Promega,Amersham International, or Boehringer Mannheim. Oligonucleotides weresynthesized by Scandinavian Gene Synthesis AB or Pharmacia Biotech andare listed in Table 2.

TABLE 2 Oligonucleotides. Name Use Sequence Pe Sequencing of pHEN1clones 5′-TTG CCT ACG GCA GCC GCT GAA-3′ (SEQ ID NO:8) My Sequencing ofpHEN1 clones 5′-TGC GGC CCC ATT CAG ATC CTC-3′ (SEQ ID NO:9) Olg1Sequencing of sbi 5′-CTC CAT ATA GTA CTT CCT TA-3′ (SEQ ID NO:10) Olg2Sequencing of sbi 5′-GAG ATT GCA TCA TTT GCT GA-3′ (SEQ ID NO:11) Olg3Sequencing of sbi 5′-GTA ACC ATA GTT AAA TGA AT-3′ (SEQ ID NO:12) Olg4Sequencing of sbi 5′-CGA TAA ATC AGC AGC ATA TG-3′ (SEQ ID NO:13) Olg5Sequencing of sbi 5′-CAA TCA CCA CAA ATT GAA AA-3′ (SEQ ID NO:14) Olg6Sequencing of sbi 5′-TGG TGC TTG TAG TGG AAA AG-3′ (SEQ ID NO:15) Olg8PCR for MAL-E fusions 5′-AGT GGA TCC ACG CAA CAA ACT TCA ACT AAG CA-3′(SEQ ID NO:16) Olg9 PCR for MAL-E fusions 5′-AAT GTC GAC AAA CTA GAG AAGATA TTT TTG A-3′ and constr. of sbi-probe (SEQ ID NO:17) Olg10 PCR forMAL-E fusions 5′-TAG GAT CCG TAC AAT CTT CTA AAG CTA AAG A-3′ andconstr. of sbi-probe (SEQ ID NO:18)

All DNA manipulations were performed using standard methods (Sambrook etal, 1989), except ligations and small scale plasmid preparations, forwhich the Ready to Go-ligation kit (Pharmacia Biotech) and Wizard™Miniprep DNA Purification systems (Promega), respectively, were usedaccording to the manufacturers' instructions. Plasmids were introducedinto E. coli and staphylococci by electrotransformation. Staphylococcalchromosomal DNA was prepared according to Lindberg et al (1972).

DNA was sequenced according to the dideoxy chain termination methodusing the Sequenase® version 2.0 DNA sequencing kit from United StatesBiochemical. Restriction sites shown in FIG. 1 were used forconstruction of subclones used in determination of the nucleotidesequence. One additional clone, pHSBB7, was made (not shown) by Bal31exonuclease digestion from the unique HindIII site in the 3′ directionof the gene, and sequenced. Different oligonucleotides were used asprimers for determining the sequence of both DNA strands (Table 2). ThePC-gene program (Intelligenetics) was used for the handling of thesequences. The EMBL, GeneBank, SWISS-protein and PIR databases weresearched for sequence-homologies.

To express the Sbi protein, two constructs were made in thepMAL-c2-vector. Primers Olg8 and 9 were used to PCR-amplify the DNAencoding full-length protein lacking the signal sequence (aa 33-436, SEQID NO:3). Primers Olg9 and 10 were used to PCR-amplify the DNA encodinga truncated version (aa 143-436, SEQ ID NO:4) lacking also the knownIgG-binding domain. The obtained PCR-products were digested with therestriction enzymes BamHI and SalI and ligated into the vector cleavedwith the same enzymes and transformed into E. coli.

Southern blots and hybridizations were performed according to Sambrooket al (1989), using a NcoI-XhoI fragment, i.e. the complete insert fromclone Ig4, (Jacobsson & Frykberg, 1995) ³²P-labelled by random primingfor detection and cloning of the sbi-gene. For detection of the sbi-genein different S. aureus strains, the PCR-fragment obtained by using Olg9and 10 (see above) was ³²P-labelled by random-priming. The hybridisationwas carried out at 50° C. and the washing at 65° C. in 0.1×SSC(20×SSC=3.0M NaCl and 0.3M Na-citrate) and 0.1% SDS.

The original clone (FIG. 1b, clone Ig4) expressing an IgG-bindingpolypeptide was earlier isolated from a shot-gun phage display librarymade from S. aureus strain 8325-4 (Jacobsson & Frykberg, 1995). Theinsert from this clone was used as a probe for identification andsubsequently for cloning of the complete gene from S. aureus strain8325-4. Chromosomal DNA was digested with PstI and XbaI, and the DNAfragments were separated by agarose gel electrophoresis followed byblotting onto a nitrocellulose filter. Hybridization with the NcoI-XhoIfragment, derived from Ig4, showed that the gene of the inventionresided on a fragment of approximately 3 kb in size. DNA fragments ofthis size were purified by agarose gel electrophoresis and cloned intothe pUC18 vector. One clone, pPX1, hybridizing with the probe wasfurther characterized by restriction enzyme analysis. FIG. 1schematically shows the 3 kb PstI-XbaI DNA fragment containing thesbi-gene and the different restriction enzymes used for subcloning.

FIG. 2 shows 1620 nt of the 3 kb PstI-XbaI DNA fragment. The codingsequence starts with a Met at nucleotide position 181 and ends with astop codon at position 1488 (SEQ ID NO:1), encoding a protein of 436amino acids (SEQ ID NO:2), including a typical signal peptide with aputative cleavage site after amino acid 29. The gene has the normalfeatures associated with a functional gene, putative promotor sequences,a possible ribosomal binding site and an inverted repeat located afterthe translation termination stop codon. A proline rich sequence,containing eight prolines repeated every fifth amino acid, starts atposition 267. Such sequences are normally found within cell wallspanning domains. However, in this case the proline rich region is notfollowed by the cell wall sorting LPXTG motif (Schneewind et al, 1995).

Example 2 Mapping of the IG-binding Domain in Protein Sbi

The library was constructed from the cloned sbi-gene essentially asdescribed (Jacobsson 10 & Frykberg, 1995). In short, the DNA from clonepPX1 (FIG. 1a) was sonicated and DNA fragments in size of approximately50-300 bp were isolated by preparative gel electrophoresis. Thefragments were made blunt-ended with T4 DNA polymerase and ligated intothe phagemid pHEN1, previously digested with Pst1, made blunt-ended, anddephosphorylated with calf intestine alkaline phosphatase. The ligationwas made using 1 μg of vector and 1 μg of DNA fragments and the mixturewas transformed into E. coli TG1. The transformants were grown overnight in LB supplied with 50 μg ampicillin ml⁻¹ and 1% glucose (w/v) andthereafter infected with helper phage R408 at a MOI of 20. After 1 hourthe culture was diluted and ampicillin added to a final concentration of50 μg ml⁻¹. After 5 hours of growth at 37° C., the bacteria werepelletted and the supernatant, containing the phages, was sterilefiltered.

The library was affinity selected against human IgG (Kabivitrum) andpositive clones were identified using labelled IgG as described(Jacobsson & Frykberg, 1995).

In order to determine the exact position of the IgG-binding domain inprotein Sbi a shot-gun phage display library was made from clone pPX1.After panning the library against immobilized human IgG, E. coli TG1cells were infected with the eluted phage and the bacteria were spreadon LA-plates containing ampicillin. Amp^(R) colonies were analysed forbinding of HRP-labelled human IgG. Positive clones were isolated and thenucleotide sequences of the different inserts were determined. As seenin FIG. 1 all clones had inserts derived from the same part of sbi,suggesting that the encoded protein has only one IgG-binding domain.From the sequences of the binding clones in FIG. 1, the minimalIgG-binding domain is deduced to consist of 52 amino acids.

Example 3 Analysis of the Immunoglobulin Specificity of Protein Sbi

To determine the specificity of protein Sbi, a phage-stock was preparedas described above from clone Ig4, encoding the IgG-binding domain ofprotein Sbi (Jacobsson & Frykberg, 1995). The stock was diluted to 10⁸phagemid particles ml⁻¹ and 100 μl was panned as described (Jacobsson &Frykberg, 1995) against human IgG, IgM, IgA, IgG3κ, IgG3λ as well as IgGfrom rat, goat, pig, cow, sheep, horse, guinea pig, dog, rabbit andchicken (Sigma), immobilized in microwells at a concentration of 50 μgml⁻¹. BSA was included as a negative control. The number of boundphagemid particles was determined as c.f.u. after infection of E. coliTG1 cells with the phage eluated at pH 2.

To analyse the Ig-binding properties of protein Sbi, clone Ig4 encodingthe IgG-binding domain, was used to produce phage-particles displayingthe binding domain on the surface. These phages were panned againstdifferent immunoglobulins and the number of binding phagemid-particleswas determined and used as a measure of the binding ability (FIG. 3).

Example 4 Protein Purification and Electrophoresis

The MAL-fusion proteins, MAL-Sbi (aa 33-436, SEQ ID NO:3) andMAL-SbiΔ(aa 143-436, SEQ ID NO:4), were purified from E. coli lysates onan amylose-resin according to the manufacturer's instructions (NewEngland Biolabs). MAL-Sbi was further purified on IgG-Sepharose®according to the manufacturer's instructions (Pharmacia Biotech).

Antibodies against protein Sbi, developed in chickens, were obtainedthrough Immunsystem AB (Uppsala, Sweden) (four immunizations of 50 μgMAL-SbiΔ-protein). The antibodies were affinity-purified on immobilizedMAL-SbiΔ-protein and labelled with Horse-radish peroxidase (HRP)(Boehringer Mannheim). Commercially available HRP-labelled chickenantibodies against protein A were ordered from Immunsystem AB.

To further characterize the protein encoded by the sbi-gene thePst1-XbaI fragment from pPX1, encoding the complete protein, was madeblunt ended and inserted into the Sma1 site in the shuttle plasmidpBR473. The construct, pShPX1, was first introduced into the restrictionnegative S. aureus strain 113 and plasmid DNA was prepared from onechloramphenicol resistant colony. The plasmid was then transferred to S.epidermidis strain 247, known to be negative in IgG-binding as allcoagulase negative staphyolococci, and chloramphenicol resistantcolonies were screened for binding of HRP-labeled IgG. All clonesanalysed had recieved IgG-binding capacity and one of the clones, SePX1,was chosen for further studies. Chromosomal DNA was prepared from thisclone, cleaved with SnaBI and HindIII and analysed by Southern blothybridization, which showed that the clone contained the sbi gene asexpected (data not shown).

The bacterial cells in over night-cultures of Newman 4 (2 ml), Cowan 1(2 ml) and S. epidermidis containing pShPX1 (40 ml) were collected bycentrifugation and washed once in PBS (phosphate buffered saline pH7.4). Newman 4- and S. epidermidis-cells were resuspensed in 40 μl PBSfollowed by addition of 40 μl 2×sample-buffer (1×buffer=125 mM Tris-HCIpH 6.8, 10% glycerol, 5% (v/v) β-mercaptoethanol, 2% SDS and 0.1%bromophenol blue) and the samples boiled for 2 minutes. To releaseprotein A, the same amount of cells in PBS was treated with lysostaphin(0.1 mg/ml, Sigma) for 5 minutes at 37° C. before addition of thesample-buffer and boiling for 2 minutes. The 1 μl of the samples wereanalyzed by SDS-PAGE using the Phast-system (Pharmacia Biotech). Alsoincluded were 1/40 μg protein A (Pharmacia Biotech), 1/20 μg MAL-Sbi(SEQ ID NO:3) and 1/20 μg MAL-SbiΔ (SEQ ID NO:4). The proteins wereblotted onto nitrocellulose-filters (Schleicher & Schuell) and proteinsdetected using HRP-labelled affinity purified rabbit anti-chicken(diluted 1/500, Sigma), anti-Sbi (10 μg/ml) or anti-protein A (15μg/ml). Bound antibodies were detected using 4-chloro-1-naphtol (Serva).

To further characterize the protein encoded by the sbi-gene, two cloneswere made in the pMAL-c2 vector, expressing the mature full lengthprotein (MAL-Sbi, aa 33-436, SEQ ID NO:3) and a truncated protein(MAL-SbiΔ, aa 143-436, SEQ ID NO:4) lacking the known IgG-bindingdomain. The purified products were analysed by SDS-PAGE and Western blottogether with commercially available protein A, cell surface extract ofS. epidermidis containing pShX1 and S. aureus Newman 4, as well as lysedNewman 4 and Cowan I cells. Three duplicate gels were blotted ontonitrocellulose-filters and the blots developed with HRP-labelled rabbitIgG for detection of protein A and Sbi. anti-protein A and anti-Sbi,respectively.

Western blot analysis of purified proteins shows that antibodies againstMAL-SbiΔ (SEQ ID NO:4) recognise both MAL-Sbi (SEQ ID NO:3)and MAL-SbiΔ(SEQ ID NO:4) but not protein A. Commercially available antibodiesagainst protein A (Immunsystem AB, Uppsala) recognise both protein A andMAL-Sbi but not Mal-SbiΔ. This means that the antibodies against proteinA recognise the IgG-binding domain in protein Sbi. The antibodiesagainst the polypeptide fragment MAL-SbiΔ of the protein Sbi arespecific for the protein Sbi. Western blot analysis of lysates of twodifferent S. aureus strains show that the antibodies against MAL-SbiΔ(SEQ ID NO:4) only recognise protein Sbi and no other staphylococcalprotein.

Example 5 Occurence of the sbi-gene in Staphylococcal Strains

Chromosomal DNA was isolated from S. aureus strains 8325-4, Cowan I,Newman 4 and Wood 46. The DNAs were digested with the enzyme HindIIIfollowed by agarose gel electrophoresis and then blotted onto anitrocellulose filter. The Southern blot shows that the sbi-gene ispresent in all four S. aureus strains tested.

Example 6 Purification of Apolipoprotein H

Purified MAL-SbiΔ (SEQ ID NO:4) was immobilised on CNBr-activatedSepharose® (Pharmacia Biotech) according to the manufacturer'sinstruction. Ten ml rat (ICN), bovine (Life Technologies) or human(Uppsala university hospital) serum was diluted 20 times in PBS andpassed over the column. After extensive washing with acetate-buffer pH5.5, the bound protein was eluted in acetate buffer pH 2.7. The eluatewas lyophilised and the protein dissolved in water.

The proteins were analysed by SDS-PAGE and blotted ontonitrocellulose-filters (Schleisher-Schuell) together with commerciallyavailable human apolipoprotein H (ICN). The proteins were detected withrabbit anti-human apolipoprotein H antibodies (Chemicon) andHRP-labelled goat anti-rabbit antibodies (Santa Cruz Biotechnology). Anidentical Western blot was treated with purified MAL-Sbi after whichHRP-labelled chicken antibodies against protein Sbi was added. Bothfilters were developed with 4-chloro-1-naphtol.

These results show that in addition to IgG, protein Sbi bindsapolipoprotein H from various mammals and that it can be used forpurification of apolipoprotein H.

Example 7 Purification of Apolipoprotein H, and Immunsation with ProteinFragments

Apolipoprotein H from human and bovine sera were also purified onSbiApoB (aa 145-267, SEQ ID NO:6). This domain is used for immunisationin different animals (rat and guinea pig) to investigate if it confersprotection against S. aureus infections. The recombinant protein SbiIgGB(31-150, SEQ ID NO:5) is also included in this immunisation study.

The Impact™ T7-system from New England Biolabs was used for constructionand purification of SbiIgGB (aa 31-150, SEQ ID NO:5) and SbiApoB (aa145-267, SEQ ID NO:6). The corresponding parts of the sbi-gene wasamplified by PCR using the oligonucleotides IgG1pr (5′-CAT GCC ATG GAAAAC ACG CAA CAA ACT TCA- 3′, i.e. SEQ ID NO:19), IgG2pr (5′-TTC TTT AGCTTT AGA AGA-3′, i.e. SEQ ID NO:20), Apolpr (5′-CAT GCC ATG GAA TCT TCTAAA GCT AAA GAA CGT- 3′, i.e. SEQ ID NO:21) and Abobin (5′-TGG CGC CACTTT CTT TTC AGC-3′, i.e. SEQ ID NO:22). The PCR-products were digestedwith NcoI and treated with T4 polynucleotide kinase and ligated into thepTYB4 vector. The vector had previously been digested with the NcoI andSmaI and treated with alkaline phosphatase. The constructions weretransformed into BL21(DE3)pLysS-cells and protein was then purifiedaccording to the manufacturer's instructions.

The purified SbiApoB was coupled to a Hitrap® affinity column (AmershamPharmacia BioTech). Bovine serum (Life Technologies) was applied ontothe column. After extensive washing with PBS (phosphate buffered saline)supplied with 0.05% Tween20, bound protein was eluted in 0.1 M glycinepH 3.0. The purified protein was analysed by SDS-PAGE andWestern-blotting and was shown to be recognised by rabbit anti-humanapolipoprotein H antibodies (Chemicon).

Example 8 Passive Immunisation with Protein Fragments

As prophylaxis or for treatment of acute infections in a patient,passive immunisation may be effected by administration of antibodiesdirected against the protein Sbi.

These antibodies can be obtained by immunisation of horses with a doseof 50-1000 μg of the polypeptide fragments SEQ ID NO:4 and/or SEQ IDNO:6 of the protein Sbi, optionally coupled to a carrier, at tree tofour separate occasions, followed by purification of the total antibodyfraction and/or the specific antibody fraction directed against thenon-IgG binding portions of the protein Sbi from serum.

In addition, antibodies can be obtained by immunisation of chickens with10-100 μg of the polypeptide fragments SEQ ID NO:4 and/or SEQ ID NO:6 ofthe protein Sbi, optionally coupled to a carrier proteins perimmunisation, at 3-4 separate occasions. The specific antibodies againstthe non-IgG binding portions of the protein Sbi can then be purifiedfrom the eggs and used for passive immunisation. Alternatively, the raweggs can be consumed to give passive protection.

Finally, antibodies can also be isolated from humans that have recoveredfrom a staphylococcal infection by purification of the total antibodyfraction and/or the specific antibody fraction directed against thenon-IgG binding portions of the protein Sbi from serum.

Although the invention has been described with regard to its preferredembodiments, which constitute the best mode presently known to theinventors, it should be understood that various changes andmodifications as would be obvious to one having the ordinary skill inthis art may be made without departing from the scope of the inventionwhich is set forth in the claims appended hereto.

References

Boyle, M. D. P. (1990). Bacterial Immunoglobulin-Binding Proteins, vols.1 and 2, London, Academic Press, Inc. (London), Ltd.

Hoogenboom, H. R., Griffiths, A. D., Johnson, K. S., Chiswell, D. J.,Hudson, P. & Winter, G. (1991). Multi-subunit proteins on the surface offilamentous phage: methodologies for displaying antibody (Fab) heavy andlight chains. Nucl Acids Res 19, 4133-4137.

Jacobsson, K. & Frykberg, L. (1995). Cloning of ligand-binding domainsof bacterial receptors by phage display. BioTechniques 18, 878-885

Jönsson, K., McDevitt, D., Homonylo McGavin, M., Patti, J. M. & Höök, M.(1995). Staphylococcus aureus expresses a major histocompatibilitycomplex class II analog. J. Biol. Chem. 270,21457-21460.

Lindberg, M., Sjöström, J.-E. & Johansson, T. (1972). Transformation ofchromosomal and plasmid characters in Staphylococcus aureus. J Bacteriol109, 844-847.

Park, P. W., Rosenbloom, J., Abrams, W. R., Rosenbloom, J. & Mecham, R.P. (1996). Molecular cloning and expression of the gene forelastin-binding protein (ebpS) in Staphylococcus aureus. J. Biol. Chem.271, 15803-15809.

Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989). Molecular Cloning: aLaboratory Manual, 2nd edn. Cold Spring Harbor, N.Y.: Cold SpringHarbour Laboratory.

Schneewind, O., Fowler, A. & Faull, K. F. (1995). Structure of cell wallanchor of surface proteins in Staphylococcus aureus. Science 268,103-106.

Uhlén, M., Guss, B., Nilsson, B., Gatenbeck, S., Philipson, L. &Lindberg, M. (1984). Complete sequence of the staphylococcal geneencoding protein A, a gene evolved through multiple duplications. J BiolChem 259, 1695-1702.

6 1 1620 DNA Staphylococcus aureus 1 agtacttcct tacttaaaat acgctgaatgttctgaatta aacgcttttt tacatagtta 60 acactagtta atctattagt taacattagttaataattag ttaatttcca tttgtattct 120 catgtgataa attctaaaag catacaataaatttaatatg taaaaagaaa gggaatacac 180 atgaaaaata aatatatctc gaagttgctagttggggcag caacaattac gttagctaca 240 atgatttcaa atggggaagc aaaagcgagtgaaaacacgc aacaaacttc aactaagcac 300 caaacaactc aaaacaacta cgtaacagatcaacaaaaag ctttttatca agtattacat 360 ctaaaaggta tcacagaaga acaacgtaaccaatacatca aaacattacg cgaacaccca 420 gaacgtgcac aagaagtatt ctctgaatcacttaaagaca gcaagaaccc agaccgacgt 480 gttgcacaac aaaacgcttt ttacaatgttcttaaaaatg ataacttaac tgaacaagaa 540 aaaaataatt acattgcaca aattaaagaaaaccctgata gaagccaaca agtttgggta 600 gaatcagtac aatcttctaa agctaaagaacgtcaaaata ttgaaaatgc ggataaagca 660 attaaagatt tccaagataa caaagcaccacacgataaat cagcagcata tgaagctaac 720 tcaaaattac ctaaagattt acgtgataaaaacaaccgct ttgtagaaaa agtttcaatt 780 gaaaaagcaa tcgttcgtca tgatgagcgtgtgaaatcag caaatgatgc aatctcaaaa 840 ttaaatgaaa aagattcaat tgaaaacagacgtttagcac aacgtgaagt taacaaagca 900 cctatggatg taaaagagca tttacagaaacaattagacg cattagttgc tcaaaaagat 960 gctgaaaaga aagtggcgcc aaaagttgaggctcctcaaa ttcaatcacc acaaattgaa 1020 aaacctaaag tagaatcacc aaaagttgaagtccctcaaa ttcaatcacc aaaagttgag 1080 gttcctcaat ctaaattatt aggttactaccaatcattaa aagattcatt taactatggt 1140 tacaagtatt taacagatac ttataaaagctataaagaaa aatatgatac agcaaagtac 1200 tactataata cgtactataa atacaaaggtgcgattgatc aaacagtatt aacagtacta 1260 ggtagtggtt ctaaatctta catccaaccattgaaagttg atgataaaaa cggctactta 1320 gctaaatcat atgcacaagt aagaaactatgtaactgagt caatcaatac tggtaaagta 1380 ttatatactt tctaccaaaa cccaacattagtaaaaacag ctattaaagc tcaagaaact 1440 gcatcatcaa tcaaaaatac attaagtaatttattatcat tctggaaata atcaatcaaa 1500 aatatcttct ctagttttac atcattttttaaataatttt cgtaacaaac cgtgattaaa 1560 aagaaccgtt gattctcaat cgaatctacggttctttttt cattttccat caattaaatg 1620 2 436 PRT Staphylococcus aureus 2Met Lys Asn Lys Tyr Ile Ser Lys Leu Leu Val Gly Ala Ala Thr Ile 1 5 1015 Thr Leu Ala Thr Met Ile Ser Asn Gly Glu Ala Lys Ala Ser Glu Asn 20 2530 Thr Gln Gln Thr Ser Thr Lys His Gln Thr Thr Gln Asn Asn Tyr Val 35 4045 Thr Asp Gln Gln Lys Ala Phe Tyr Gln Val Leu His Leu Lys Gly Ile 50 5560 Thr Glu Glu Gln Arg Asn Gln Tyr Ile Lys Thr Leu Arg Glu His Pro 65 7075 80 Glu Arg Ala Gln Glu Val Phe Ser Glu Ser Leu Lys Asp Ser Lys Asn 8590 95 Pro Asp Arg Arg Val Ala Gln Gln Asn Ala Phe Tyr Asn Val Leu Lys100 105 110 Asn Asp Asn Leu Thr Glu Gln Glu Lys Asn Asn Tyr Ile Ala GlnIle 115 120 125 Lys Glu Asn Pro Asp Arg Ser Gln Gln Val Trp Val Glu SerVal Gln 130 135 140 Ser Ser Lys Ala Lys Glu Arg Gln Asn Ile Glu Asn AlaAsp Lys Ala 145 150 155 160 Ile Lys Asp Phe Gln Asp Asn Lys Ala Pro HisAsp Lys Ser Ala Ala 165 170 175 Tyr Glu Ala Asn Ser Lys Leu Pro Lys AspLeu Arg Asp Lys Asn Asn 180 185 190 Arg Phe Val Glu Lys Val Ser Ile GluLys Ala Ile Val Arg His Asp 195 200 205 Glu Arg Val Lys Ser Ala Asn AspAla Ile Ser Lys Leu Asn Glu Lys 210 215 220 Asp Ser Ile Glu Asn Arg ArgLeu Ala Gln Arg Glu Val Asn Lys Ala 225 230 235 240 Pro Met Asp Val LysGlu His Leu Gln Lys Gln Leu Asp Ala Leu Val 245 250 255 Ala Gln Lys AspAla Glu Lys Lys Val Ala Pro Lys Val Glu Ala Pro 260 265 270 Gln Ile GlnSer Pro Gln Ile Glu Lys Pro Lys Val Glu Ser Pro Lys 275 280 285 Val GluVal Pro Gln Ile Gln Ser Pro Lys Val Glu Val Pro Gln Ser 290 295 300 LysLeu Leu Gly Tyr Tyr Gln Ser Leu Lys Asp Ser Phe Asn Tyr Gly 305 310 315320 Tyr Lys Tyr Leu Thr Asp Thr Tyr Lys Ser Tyr Lys Glu Lys Tyr Asp 325330 335 Thr Ala Lys Tyr Tyr Tyr Asn Thr Tyr Tyr Lys Tyr Lys Gly Ala Ile340 345 350 Asp Gln Thr Val Leu Thr Val Leu Gly Ser Gly Ser Lys Ser TyrIle 355 360 365 Gln Pro Leu Lys Val Asp Asp Lys Asn Gly Tyr Leu Ala LysSer Tyr 370 375 380 Ala Gln Val Arg Asn Tyr Val Thr Glu Ser Ile Asn ThrGly Lys Val 385 390 395 400 Leu Tyr Thr Phe Tyr Gln Asn Pro Thr Leu ValLys Thr Ala Ile Lys 405 410 415 Ala Gln Glu Thr Ala Ser Ser Ile Lys AsnThr Leu Ser Asn Leu Leu 420 425 430 Ser Phe Trp Lys 435 3 404 PRTStaphylococcus aureus 3 Thr Gln Gln Thr Ser Thr Lys His Gln Thr Thr GlnAsn Asn Tyr Val 1 5 10 15 Thr Asp Gln Gln Lys Ala Phe Tyr Gln Val LeuHis Leu Lys Gly Ile 20 25 30 Thr Glu Glu Gln Arg Asn Gln Tyr Ile Lys ThrLeu Arg Glu His Pro 35 40 45 Glu Arg Ala Gln Glu Val Phe Ser Glu Ser LeuLys Asp Ser Lys Asn 50 55 60 Pro Asp Arg Arg Val Ala Gln Gln Asn Ala PheTyr Asn Val Leu Lys 65 70 75 80 Asn Asp Asn Leu Thr Glu Gln Glu Lys AsnAsn Tyr Ile Ala Gln Ile 85 90 95 Lys Glu Asn Pro Asp Arg Ser Gln Gln ValTrp Val Glu Ser Val Gln 100 105 110 Ser Ser Lys Ala Lys Glu Arg Gln AsnIle Glu Asn Ala Asp Lys Ala 115 120 125 Ile Lys Asp Phe Gln Asp Asn LysAla Pro His Asp Lys Ser Ala Ala 130 135 140 Tyr Glu Ala Asn Ser Lys LeuPro Lys Asp Leu Arg Asp Lys Asn Asn 145 150 155 160 Arg Phe Val Glu LysVal Ser Ile Glu Lys Ala Ile Val Arg His Asp 165 170 175 Glu Arg Val LysSer Ala Asn Asp Ala Ile Ser Lys Leu Asn Glu Lys 180 185 190 Asp Ser IleGlu Asn Arg Arg Leu Ala Gln Arg Glu Val Asn Lys Ala 195 200 205 Pro MetAsp Val Lys Glu His Leu Gln Lys Gln Leu Asp Ala Leu Val 210 215 220 AlaGln Lys Asp Ala Glu Lys Lys Val Ala Pro Lys Val Glu Ala Pro 225 230 235240 Gln Ile Gln Ser Pro Gln Ile Glu Lys Pro Lys Val Glu Ser Pro Lys 245250 255 Val Glu Val Pro Gln Ile Gln Ser Pro Lys Val Glu Val Pro Gln Ser260 265 270 Lys Leu Leu Gly Tyr Tyr Gln Ser Leu Lys Asp Ser Phe Asn TyrGly 275 280 285 Tyr Lys Tyr Leu Thr Asp Thr Tyr Lys Ser Tyr Lys Glu LysTyr Asp 290 295 300 Thr Ala Lys Tyr Tyr Tyr Asn Thr Tyr Tyr Lys Tyr LysGly Ala Ile 305 310 315 320 Asp Gln Thr Val Leu Thr Val Leu Gly Ser GlySer Lys Ser Tyr Ile 325 330 335 Gln Pro Leu Lys Val Asp Asp Lys Asn GlyTyr Leu Ala Lys Ser Tyr 340 345 350 Ala Gln Val Arg Asn Tyr Val Thr GluSer Ile Asn Thr Gly Lys Val 355 360 365 Leu Tyr Thr Phe Tyr Gln Asn ProThr Leu Val Lys Thr Ala Ile Lys 370 375 380 Ala Gln Glu Thr Ala Ser SerIle Lys Asn Thr Leu Ser Asn Leu Leu 385 390 395 400 Ser Phe Trp Lys 4294 PRT Staphylococcus aureus 4 Val Gln Ser Ser Lys Ala Lys Glu Arg GlnAsn Ile Glu Asn Ala Asp 1 5 10 15 Lys Ala Ile Lys Asp Phe Gln Asp AsnLys Ala Pro His Asp Lys Ser 20 25 30 Ala Ala Tyr Glu Ala Asn Ser Lys LeuPro Lys Asp Leu Arg Asp Lys 35 40 45 Asn Asn Arg Phe Val Glu Lys Val SerIle Glu Lys Ala Ile Val Arg 50 55 60 His Asp Glu Arg Val Lys Ser Ala AsnAsp Ala Ile Ser Lys Leu Asn 65 70 75 80 Glu Lys Asp Ser Ile Glu Asn ArgArg Leu Ala Gln Arg Glu Val Asn 85 90 95 Lys Ala Pro Met Asp Val Lys GluHis Leu Gln Lys Gln Leu Asp Ala 100 105 110 Leu Val Ala Gln Lys Asp AlaGlu Lys Lys Val Ala Pro Lys Val Glu 115 120 125 Ala Pro Gln Ile Gln SerPro Gln Ile Glu Lys Pro Lys Val Glu Ser 130 135 140 Pro Lys Val Glu ValPro Gln Ile Gln Ser Pro Lys Val Glu Val Pro 145 150 155 160 Gln Ser LysLeu Leu Gly Tyr Tyr Gln Ser Leu Lys Asp Ser Phe Asn 165 170 175 Tyr GlyTyr Lys Tyr Leu Thr Asp Thr Tyr Lys Ser Tyr Lys Glu Lys 180 185 190 TyrAsp Thr Ala Lys Tyr Tyr Tyr Asn Thr Tyr Tyr Lys Tyr Lys Gly 195 200 205Ala Ile Asp Gln Thr Val Leu Thr Val Leu Gly Ser Gly Ser Lys Ser 210 215220 Tyr Ile Gln Pro Leu Lys Val Asp Asp Lys Asn Gly Tyr Leu Ala Lys 225230 235 240 Ser Tyr Ala Gln Val Arg Asn Tyr Val Thr Glu Ser Ile Asn ThrGly 245 250 255 Lys Val Leu Tyr Thr Phe Tyr Gln Asn Pro Thr Leu Val LysThr Ala 260 265 270 Ile Lys Ala Gln Glu Thr Ala Ser Ser Ile Lys Asn ThrLeu Ser Asn 275 280 285 Leu Leu Ser Phe Trp Lys 290 5 120 PRTStaphylococcus aureus 5 Glu Asn Thr Gln Gln Thr Ser Thr Lys His Gln ThrThr Gln Asn Asn 1 5 10 15 Tyr Val Thr Asp Gln Gln Lys Ala Phe Tyr GlnVal Leu His Leu Lys 20 25 30 Gly Ile Thr Glu Glu Gln Arg Asn Gln Tyr IleLys Thr Leu Arg Glu 35 40 45 His Pro Glu Arg Ala Gln Glu Val Phe Ser GluSer Leu Lys Asp Ser 50 55 60 Lys Asn Pro Asp Arg Arg Val Ala Gln Gln AsnAla Phe Tyr Asn Val 65 70 75 80 Leu Lys Asn Asp Asn Leu Thr Glu Gln GluLys Asn Asn Tyr Ile Ala 85 90 95 Gln Ile Lys Glu Asn Pro Asp Arg Ser GlnGln Val Trp Val Glu Ser 100 105 110 Val Gln Ser Ser Lys Ala Lys Glu 115120 6 123 PRT Staphylococcus aureus 6 Ser Ser Lys Ala Lys Glu Arg GlnAsn Ile Glu Asn Ala Asp Lys Ala 1 5 10 15 Ile Lys Asp Phe Gln Asp AsnLys Ala Pro His Asp Lys Ser Ala Ala 20 25 30 Tyr Glu Ala Asn Ser Lys LeuPro Lys Asp Leu Arg Asp Lys Asn Asn 35 40 45 Arg Phe Val Glu Lys Val SerIle Glu Lys Ala Ile Val Arg His Asp 50 55 60 Glu Arg Val Lys Ser Ala AsnAsp Ala Ile Ser Lys Leu Asn Glu Lys 65 70 75 80 Asp Ser Ile Glu Asn ArgArg Leu Ala Gln Arg Glu Val Asn Lys Ala 85 90 95 Pro Met Asp Val Lys GluHis Leu Gln Lys Gln Leu Asp Ala Leu Val 100 105 110 Ala Gln Lys Asp AlaGlu Lys Lys Val Ala Pro 115 120

What is claimed is:
 1. Isolated or purified protein expressed by abacterium of the genus Staphylococcus aureus or an immunogenic orantigenic polypeptide fragment of said protein comprising at least 15amino acid residues other than the 84 aa fragment at the position38-121, which protein comprises the amino acid sequence SEQ ID NO: 2 MetLys Asn Lys Tyr Ile Ser Lys Leu Leu Val Gly1               5                   10 Ala Ala Thr Ile Thr Leu Ala ThrMet Ile Ser Asn                 15          20 Gly Glu Ala Lys Ala SerGlu Asn Thr Gln Gln Thr 25                  30                  35 SerThr Lys His Gln Thr Thr Gln Asn Asn Tyr Val            40                  45 Thr Asp Gln Gln Lys Ala Phe Tyr GlnVal Leu His     50                  55                  60 Leu Lys GlyIle Thr Glu Glu Gln Arg Asn Gln Tyr                65                  70 Ile Lys Thr Leu Arg Glu His ProGlu Arg Ala Gln         75                  80 Glu Val Phe Ser Glu SerLeu Lys Asp Ser Lys Asn 85                  90                  95 ProAsp Arg Arg Val Ala Gln Gln Asn Ala Phe Tyr            100                 105 Asn Val Leu Lys Asn Asp Asn Leu ThrGlu Gln Glu     110             115                     120 Lys Asn AsnTyr Ile Ala Gln Ile Lys Glu Asn Pro                125                 130 Asp Arg Ser Gln Gln Val Trp ValGlu Ser Val Gln         135                 140 Ser Ser Lys Ala Lys GluArg Gln Asn Ile Glu Asn 145                 150                 155 AlaAsp Lys Ala Ile Lys Asp Phe Gln Asp Asn Lys            160                 165 Ala Pro His Asp Lys Ser Ala Ala TyrGlu Ala Asn     170                 175                 180 Ser Lys LeuPro Lys Asp Leu Arg Asp Lys Asn Asn                185                 190 Arg Phe Val Glu Lys Val Ser IleGlu Lys Ala Ile         195                 200 Val Arg His Asp Glu ArgVal Lys Ser Ala Asn Asp 205                 210                 215 AlaIle Ser Lys Leu Asn Glu Lys Asp Ser Ile Glu            220                 225 Asn Arg Arg Leu Ala Gln Arg Glu ValAsn Lys Ala     230                 235                 240 Pro Met AspVal Lys Glu His Leu Gln Lys Gln Leu                245                 250 Asp Ala Leu Val Ala Gln Lys AspAla Glu Lys Lys         255                 260 Val Ala Pro Lys Val GluAla Pro Gln Ile Gln Ser 265                 270                 275 ProGln Ile Glu Lys Pro Lys Val Glu Ser Pro Lys            280                 285 Val Glu Val Pro Gln Ile Gln Ser ProLys Val Glu     290                 295                 300 Val Pro GlnSer Lys Leu Leu Gly Tyr Tyr Gln Ser                305                 310 Leu Lys Asp Ser Phe Asn Tyr GlyTyr Lys Tyr Leu         315                 320 Thr Asp Thr Tyr Lys SerTyr Lys Glu Lys Tyr Asp 325                 330                 335 ThrAla Lys Tyr Tyr Tyr Asn Thr Tyr Tyr Lys Tyr            340                 345 Lys Gly Ala Ile Asp Gln Thr Val LeuThr Val Leu     350                 355                 360 Gly Ser GlySer Lys Ser Tyr Ile Gln Pro Leu Lys                365                 370 Val Asp Asp Lys Asn Gly Tyr LeuAla Lys Ser Tyr         375                 380 Ala Gln Val Arg Asn TyrVal Thr Glu Ser Ile Asn 385                 390                 395 ThrGly Lys Val Leu Tyr Thr Phe Tyr Gln Asn Pro            400                 405 Thr Leu Val Lys Thr Ala Ile Lys AlaGln Glu Thr     410                 415                 420 Ala Ser SerIle Lys Asn Thr Leu Ser Asn Leu Leu                425                 430 Ser Phe Trp Lys.         435


2. An immunogenic composition comprising the protein or polypeptideaccording to claim 1.